Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. They are subject to highly diverse, abundant and evolutionarily conserved posttranslational modifications. Disruption of tubulin modification levels and patterns leads to cancers, neuropathologies and defective axonal regeneration. An essential aspect of deciphering the tubulin code is to understand how the code is written i.e. the mechanism of the enzymes that introduce these modifications and how cooperation and competition between these enzymes gives rise to the complex microtubule modification patterns observed in cells. Specifically we aim (1) to determine high-resolution structures of key tubulin modification enzymes in isolation as well as in complex with the microtubule to understand their substrate specificity and catalytic mechanism; (2) to map tubulin modification sites for all modification enzymes; (3) to investigate the biochemical interplay between tubulin modification enzymes and how this gives rise to temporally and spatially regulated modification patterns. This project leverages our ability to make unmodified and recombinant single-isoform engineered human tubulin and coupled with our expertise with an array of structural techniques (X-ray crystallography, cryo-EM and SAXS), high-resolution mass spectrometry, classical kinetics and single molecule fluorescence will answer fundamental questions about the mechanism and regulation of tubulin modification enzymes. We have made significant progress towards these objectives. We determined the first three dimensional structure of a tubulin glycylase, TTLL3 and identified two functionally essential architectural elements unique to initiating glycylase and missing from elongating glycylases of the TTLL family. Moreover, we showed that TTLL3 competes with the glutamylase TTLL7 for overlapping modification sites on tubulin, providing a molecular basis for the anti-correlation between these modifications observed in vivo. Thus, our recent results illustrate with purified components how a combinatorial tubulin code can arise through the intersection of activities of TTLL enzymes. We have also extended our studies towards TTLL5 to understand how this enzyme recognizes a non-tubulin target, the retinitis pigmentosa GTPase regulator (RPGR). RPGR localizes to the connecting cilium of photoreceptors and we have previously shown that its glutamylation by TTLL5 is critical for its function in photoreceptors. In its absence, opsin transport is compromised and the TTLL5 mutant mouse develops slow photoreceptor degeneration.